System and method for diagnosis and treatment of neuropsychiatric disorders

ABSTRACT

An assay for a GCH1 allele and associated genotype for the screening, prediction, diagnosis, prognosis, treatment and treatment response of psychiatric, neuropsychiatric, and neurological disorders, such as schizophrenia, schizoaffective disorder and bipolar disorder, and for defining treatments of such disorders. The presence of a variant in the GCH1 gene, alone or in conjunction with a measurement of low or altered biopterin, or altered BH4 system measures, is used to screen or diagnose subjects at risk for developing a psychiatric, neuropsychiatric, or neurological disorder. The genetic assay, with or without a biopterin or BH4 system assay, may also be used to determine treatment regimens. For subjects with an impaired BH4 system, treatments to increase or normalize biopterin, BH4, or the BH4 system can also be used, such as BH4 supplementation, lithium treatment, phenylalanine treatment, or other treatments and therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/332,390, filed on Oct. 24, 2016, which was a continuation ofU.S. patent application Ser. No. 14/069,847, filed on Nov. 1, 2013,which was a continuation of U.S. patent application Ser. No. 13/323,413,filed on Dec. 12, 2011, which was a continuation of U.S. patentapplication Ser. No. 11/873,971, filed on Oct. 17, 2007, now U.S. Pat.No. 8,076,075, which claimed priority to U.S. Provisional ApplicationSer. No. 60/829,856, filed Oct. 17, 2006.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.MH067941 and MH066883 of the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to psychiatric and neuropsychiatricdisorders, such as schizophrenia, schizoaffective disorder, bipolardisorder, and Alzheimer's disease and, more particularly, to the role ofGTP cyclohydrolase I gene, GTP cyclohydrolase (GTPCH), biopterins, andtetrahydrobiopterin in the detection, diagnosis, prognosis and treatmentof such disorders.

2. Description of the Related Art

Schizophrenia (SZ) and schizoaffective disorder (SaD) are among the mostcommon forms of mental illness, and have large genetic and heritablecomponents, indicated by studies showing increased risk among firstdegree relatives, and concordance between mono- and dizygotic twins. Thegenetic components of SZ and SaD appear to involve multiple genes.Individuals with these psychiatric disorders can display an overlappingrange of symptoms and there appears to be increased prevalence of SZ inthe families of SaD sufferers, and vice versa. There have also beenreports of shared genetic susceptibility loci for these disorders.

Tetrahydrobiopterin (BH4) is a vital cofactor maintaining availabilityof the amine neurotransmitters, dopamine (DA), noradrenaline (NA), andserotonin (5-HT). BH4 is also involved in regulating the synthesis ofnitric oxide (NO) by nitric oxide synthases (NOS), and stimulating andmodulating the glutamatergic system. In the central nervous system(CNS), BH4 has also been shown to stimulate the release of DA, 5-HT andglutamate, as well as regulating the expression of tyrosine hydroxylaseat nerve terminals. Plasma total biopterins level (biopterin) is ameasure of BH4 (approximately 80-90% in the form of BH4) and arecorrelated with CNS biopterin levels.

Conventional methods do not rely on a genetically based method forassessing the presence or risk of schizophrenia or schizoaffectivedisorder using the GTP cyclohydrolase I (GCH1) gene alone or inconjunction with a biochemical assay. These methods also fail todisclose a method of treating psychiatric and neuropsychiatric diseases,such as schizophrenia, by addressing genetic deficiencies in the GCH1gene and/or in the BH4 system. For example, there is currently no usefulgenetic test for determining subjects that are at-risk for developingschizophrenia and, as a result, treatment approaches have limitedsuccess.

BRIEF SUMMARY OF THE INVENTION

It is therefore a principal object and advantage of the presentinvention to provide a system and method for assessing the presence orrisk or severity or progression or prognosis of certain psychiatric andneuropsychiatric diseases, and neurological disorders.

It is an additional object and advantage of the present invention toprovide a system and method for testing for the presence or risk ofpsychiatric, neuropsychiatric, and neurological disorders.

It is a further object and advantage of the present invention to providea system and method for the determination of treatment of psychiatricand neuropsychiatric diseases, and neurological disorders.

It is a further object and advantage of the present invention to providea treatment for psychiatric and neuropsychiatric diseases, andneurological disorders.

In accordance with the foregoing objects and advantages, the presentinvention provides for genetic testing of GCH1, either as a stand-alonetest, or in conjunction with assay of biopterin, BH4, and/or BH4 systemmeasures for the assessment of psychiatric and neuropsychiatricdiseases, such as schizophrenia, either alone or in conjunction withassays of other genes. Symptom scales, behavioral measures,physiological testing, biological or molecular or genetic testing, andimaging analyses, will also be used where appropriate, in conjunctionwith genetic testing, assay of biopterin, BH4, or BH4 system measures.In particular, a GTP cyclohydrolase I (GCH1) gene variant (nucleotidevariant −959nt G/A: rs10137071, NCBI dbSNP database) “A” allele, ispresent in a much larger than expected proportion of psychiatricpatients (schizophrenics (SZ) and schizoaffective disorder (SaD)), thanin healthy people. For example, the odds ratio of having the GCH1variant genotype was over five fold higher in SZ and SaD patients whencompared to healthy control subjects. The risk of having a psychiatricdisorder is therefore multiplied by five times for people who carry the“A/A” variant.

The present invention is based on an assay of GCH1 genotype, separatelyand/or in conjunction with assay of biopterin or other pterins in blood,plasma, serum, CSF, or other fluids or tissues, and/or assays of otherBH4 system measures, and is useful in the prediction, diagnosis andprognosis of psychiatric disorders, and for defining treatments. Inaddition, treatments such as BH4, biopterin, other pterin species,phenylalanine, lithium, or other treatments designed or known toincrease biopterin or BH4 (or normalize the BH4 system) in persons witha variant GCH1 genotype (and thus to alleviate biopterin deficit or toprevent a deficit in persons at-risk for the disorders), may beadministered to provide a therapeutic or preventative response ortreatment in patients with the disorders or at-risk for developing thedisorders. The assay of GCH1 genotype, with or without biopterin or BH4assay, may also be used to determine antipsychotic or mood stabilizermedication, as well as other treatment requirements. For subjects withan impaired BH4 system, treatments to increase BH4 or normalize BH4 canbe used, such as supplementation with BH4 (or other pterin moleculespecies), lithium treatment, phenylalanine treatment, or other usefultreatments, such as ECT.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be more fully understood and appreciated byreading the following Detailed Description in conjunction with theaccompanying drawings, in which:

FIGS. 1A and B are schematics of biosynthesis pathways of BH4 and itsroles in the hydroxylation of the aromatic amino acids to the amineneurotransmitters, nitric oxide (also as a cofactor) and its roles instimulation and modulation of neurotransmitter synthesis and release.

FIG. 2 is a chart illustrating the change in biopterin levels withlithium as a covariate.

FIG. 3 is a graph of the ranked plasma biopterin levels and GCH1genotype within test diagnostic groups.

FIG. 4 is a graph of the plasma biopterin values and GCH1 genotypes forthe test subject data.

FIG. 5 illustrates the sequence of the “g” allele of the GCH1 variant ofthe present invention (SEQ ID NO:1), the sequence of the “a” allele ofthe GCH1 variant of the present invention (SEQ ID NO:2), the sequence ofthe forward primer according to the present invention (SEQ ID NO:3), thesequence of the reverse primer according to the present invention (SEQID NO:4).

FIG. 6 is a graph illustrating leukocyte GCH1 expression in the SZ Aallele group when compared to SZ and BpD G/G subjects.

FIG. 7 is a graph illustrating leukocyte gene expression for BpDsubjects.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a screening (including populationscreening), risk assessment, prognostic and diagnostic test for SZ andSaD, and, if genetic and biochemical testing is combined, a screening,risk assessment, diagnostic and prognostic test for bipolar disorder(BpD). In addition, the present invention provides for treatments suchas biopterin, BH4 or other pterin species (which have been successfullyused to treat autism and other disorders in children, and depression andother disorders in adults, and so safe therapeutic doses have previouslybeen determined), other treatments that increase BH4 (such as lithium)or biopterin, treatments which alleviate the biopterin deficit or thepotential biopterin deficit in those at risk, or treatments which may beused to normalize BH4. The present invention is therefore be used toallow determinations of treatment use, and to provide treatments foralleviating psychiatric, neuropsychiatric, or neurological symptoms. Thepresent invention will also provide for early detection and prophylacticor preventative treatment for those at risk for developing thedisorders, such as children and adolescents, or others at risk.

The present invention further allows for assessment of GCH1 genotypealone, or in conjunction with assessment of one or more of biopterin,BH4, neopterin or other pterins, GCH1 RNA, epigenetic modificationsincluding DNA methylation, GTPCH protein levels, GTPCH enzyme activityand/or the BH4 system, that can be used to determine treatment needs.The present invention may also be used to diagnose and/or prognoseand/or determine treatment, for other disorders including but notlimited to those described herein.

The term “BH4 system” includes biopterin (total biopterins or individualbiopterin species), BH4, neopterin (total neopterins or individualneopterin, such as dihydroneopterin trophosphate), other pterin species,and/or the genes, RNA, proteins, and enzymes that form the pathways ofBH4 biosynthesis and metabolism (including the de novo synthesis,regeneration, and salvage pathways, and cofactor reaction pathways). Theterm also includes genes, RNA, proteins, enzymes and metabolites thatcan directly and/or indirectly influence the biosynthesis of BH4 and/orthe pathways of BH4 biosynthesis and metabolism, including the de novosynthesis, regeneration, and salvage pathways, and cofactor reactionpathways and associated genes, including the genes GCH1 GCHFR, PTS, SPR,PCBD, QDPR, NOS(I,II,III), PAH, TH, TPH, DHFR, MTHFR, AKR1B1, AKR1C3,AKR1C1, CBR1, NR4A2, CTF1, IL6, MTPN, LIF, CNTF, PRKG2, and the genes,proteins, enzymes, and/or metabolites relating to GSK3 alpha and/orbeta, NURR1 and/or Nur77, Nurr1 and/or NOR1, IMPase and/or inositolphosphate-1-phosphotase (IPP), v-akt murine thymoma viral oncogenehomolog 1 (AKT1), AKT/PKB, AKT2, AKT3, etc. The term BH4 system alsoincludes the promoters, enhancers, supressors, and other regulatoryregions, regulatory RNAs (such as microRNA) of “BH4 system” genes. Theterm BH4 system also includes epigenetic regulation such as methylationof BH4 system genes, and acetylation and/or ubiquitylation ofchromosome-associated proteins such as histones.

The term “assay of BH4 system measures” includes assays and/ormeasurements of biopterin or BH4 or BH4 system DNA, RNA, genes, miRNA,methylation, acetylation, ubiquitylation, proteins, enzymes, and/ormetabolites, other pterin species, GCH1 RNA, GTPCH protein, and/or GTPCHenzyme activity (and the RNA, protein and enzyme activities of the otherBH4 system genes, including GCH1 GCHFR, PTS, SPR, PCBD, QDPR,NOS(I,II,III), PAH, TH, TPH, DHFR, MTHFR, AKR1B1, AKR1C3, AKR1C1, CBR1,NR4A2, CTF1, MTPN, LIF, CNTF, PRKG2), whether in plasma, serum, blood,CSF, urine, or saliva, as well as other tissues, fluids, cells, organsor substances. Assays of BH4 system measures, e.g., assays ofbiopterins, BH4, neopterins, and pterin species, include the use of HPLCmethods. Assay of BH4 system measures also includes use of massspectrometry methods, immunoassay, radioimmunoassay,immunohistochemistry, MRS, spectroscopy methods, radio-tracer assayand/or imaging methods, electrophoresis methods, molecular biologymethods, including PCR and real time PCR, genetic sequencing methods,SNP assay methods, biochemical measurement methods, enzyme activitymeasurement methods, protein measurement methods, DNA, RNA, miRNA orprotein microarray assay methods, other DNA, RNA and protein multiplexassay methods, and other assay and measurement methods known to thoseskilled in the art.

The term “psychiatric disorders” includes but is not limited to: SZ,SaD, BpD, mood disorders and personality disorders, unipolar depressivedisorder, psychotic disorders, major depressive and other depressivedisorders, other affective disorders, attention deficit disorder,delusional disorder, anxiety disorders, obsessive compulsive disorder,paranoid schizotypal or schizoid personality.

The term “neuropsychiatric disorders” includes, but is not limited to,neurological, neuropsychiatric and neurodegenerative disorders,including Alzheimer's disease, Pick's disease, Parkinson's disease,Huntington's disease, multiple sclerosis, Wilson's disease,Creutzfeldt-Jakob disease and other disorders of the central nervoussystem and the peripheral nervous system including abnormal orheightened pain sensitivity, as well as movement disorders includingdyskinesias, dystonias and akathisias, autism, Asperger's syndrome andspectrum disorders, and autism spectrum disorders, and also dementias,cognitive status and decline, intellectual status and decline, learningor memory status decline, and other intellectual disabilities anddisorders.

The term “patient” includes human subjects, including fetuses, as wellas includes organ, tissue, cell, fluid, DNA, RNA, protein, chemicaland/or material samples from patients and other organisms (e.g.,animals). The term model includes animal models of diseases and states,and chimeric models.

The term “assay of GCH1 genotype” includes, but is not limited to,determination of the −959nt G/A: rs10137071 GCH1 genotype and/or one ormore other genetic change affecting GCH1, as well as genetic orepigenetic difference such as a nucleotide variant affecting the GCH1gene or a copy number polymorphism or duplication, deletion or othermutation or change, in GCH1 or its promoters, enhancers, suppressors andother regulatory regions, and any other RNA species, e.g., regulatoryRNAs such as miRNAs that regulate GCH1 gene expression. The term “assayof GCH1 genotype” also includes determination of epigenetic regulationsuch as DNA methylation, and/or acetylation and/or ubiquitylation ofchromosome-associated proteins such as histones, affecting the GCH1gene.

The term “normalize their BH4 system levels” includes treatment tonormalize biopterin(s), other pterin species levels, BH4 levels, GCH1RNA, GTPCH protein, tyrosine hydroxylase, tryptophan hydroxylase levels,the activities of the BH4 system enzymes, and/or the function of the BH4system. The term “normalize their BH4 system levels” also includestreatment to normalize or treat or alter or supplement, the sequencesand/or function of BH4 system genes and/or their regulatory regions orepigenetic regulators, including by gene therapy methods. For example,treatment may occur by introduction of nucleic acids to provide adequateand/or functional and/or normal gene functioning, to those in need oftreatment, including those with, or at risk of developing a psychiatricor neuropsychiatric or neurological disorder. One or more nucleic acidscan be provided or introduced to patient's bodies, cells, organs,tissues or fluids. Nucleic acids to be provided can include for exampleone, or more than one different, or several different, GCH1 nucleicacids, such as an oligonucleotide incorporating the gene variant(nucleotide variant −959nt G/A: rs10137071, NCBI dbSNP database) “G”nucleotide, using methods known to those skilled in the art.

The term “a GCH1 variant” includes the −959nt guanine/adenine (G/A):rs10137071 GCH1, “A” allele and/or “G/A” or “A/A” genotype and/or othergenetic change(s) affecting GCH1.

The term “useful treatments” includes treatment with pterin(s) and/orother molecules, including BH4, biopterin, dihydrobiopterin,sepiapterin, sapropterin, dihydroneopterin triphosphate or otherneopterins, Lithium (Li) or Li-based medications, phenylalanine,aspartame, sapropterin dihydrochloride, peptide molecules containingresidues such as phenylalanine, tyrosine, and/or tryptophan, inhibitorsof glycogen synthase kinase 3 (beta and/or alpha) expression, enzymeactivity, or catalysis, inositol phosphate-1-phosphotase (IPP)expression, enzyme activity, or catalysis, Inosotiol monophosphate(IMPase) expression, enzyme activity, or catalysis, promoters orinhibitors of AKT expression, enzyme activity, or catalysis, promotersof glycogen synthase kinase 3 (beta and/or alpha) phosphorylation,administration of electrical stimulation and/or electroconvulsivetherapy (ECT), transcranial magnetic stimulation (TMS), electrical brainstimulation, deep brain stimulation and/or other electrical, magnetic orradiowave general or focally targeted brain stimulation, and/or inositoldepletion treatments. Useful treatments may also include neuroleptic(s)and/or other antipsychotic and/or mood stabilizer(s) and/or otherpsychotropic(s) and/or other medication(s)

The term “low biopterin” includes, but is not limited to, biopterinlevels (or other BH4 system measures) that are either lower than themean, median, mode, “normal” level, or reference range level for controlor healthy subject groups (groups can be defined by factors includingbut not limited to, ethnicity, race, age, gender, BMI, weight,nutritional status, health status). The term “low biopterin” alsoincludes levels that are outside of levels needed for optimal or normalbrain, CNS, biological, biochemical, and/or physiological function.Subject biopterin level can also be adjusted to reflect the influence ofmodifiers of biopterin level, BH4 level, and/or Phe levels, such as thatdue to nicotine, or to treatments such as lithium, ECT, TMS, deep brainstimulation, or other treatments or therapies. Low biopterin can also bedefined as a range or level compared to different population(s) and/orgroup norms and/or arbitrary cutoffs. Abnormal biopterin levels can alsoinclude elevated or “high” BH4 system measures.

The definitions, such as those described above, can also be utilized todefine “low” biopterin, BH4 or other measures, such as other pterinspecies, GCH1 RNA, GTPCH protein, and/or GTPCH enzyme activity, or otherBH4 system measures. Definitions can also be based upon comparisons(e.g., with reference ranges) and/or statistical descriptions includingthose such as percentiles, standard deviations, confidence intervals,standard errors, etc.

The present invention is based on the fact that a GTP cyclohydrolase I(GCH1) homozygous gene variant (nucleotide variant −959nt G/A:rs10137071, NCBI dbSNP database) “A” allele, is present in a much largerthan expected proportion of psychiatric patients (schizophrenics (SZ)and schizoaffective disorder (SaD)), than in healthy people. Forexample, the odds ratio of having the GCH1 variant genotype was nearlyfive fold higher in SZ and SaD patients when compared to healthy controlsubjects. The risk of having a psychiatric disorder is thereforemultiplied by five times for people who carry the “A/A” variant.

Patients with the GCH1 “A” allele and, in particular, the “A/A”genotype, have decreased fasting blood plasma levels of a totalbiopterins, known as “biopterin,” that is from, and is a measure oftetrahydrobiopterin (BH4). BH4 is a vital cofactor that is required forthe maintenance of neurotransmitters in the brain and periphery, andthese neurotransmitters have been implicated in psychiatric disorders.The GTP cyclohydrolase I (GCH1) gene encodes GTP-cyclohydrolase (GTPCH),the first enzyme in BH4 biosynthesis. This finding directly links theGCH1 gene “A” variant to the increased risk for these psychiatricdisorders.

As the GCH1 “A” variant is a risk allele for SZ, SaD, and BpD, havingthe GCH1 variant genotype has biological consequences that likelyresults in decreased GCH1 gene expression or altered GCH1 splicing, andthus the decreased biopterin level observed in patients with the variant“A” allele. As GCH1 encodes an enzyme that is required for BH4biosynthesis (called GTPCH), lower or altered GCH1 expression likelyresults in the plasma biopterin deficit in SZ and SaD populations. SZpatients have lower GCH1 gene expression than healthy control subjects,and SZ subjects with the GCH1 “A” allele have lower GCH1 expression thanSZ without the “A” allele, and thus are likely to exhibit a similarbrain biopterin deficit (or BH4 system deficit) that will result in thedysregulation of neurotransmitters, such as dopamine, noradrenaline,serotonin and the glutamatergic system, nitric oxide, and with result inan SZ, SaD, or other psychiatric or neuropsychiatric disorder orneurological disorder phenotype. Similarly, GCH1 expression is lower in“A” allele BpD patients compared to BpD without the A allele, and theseBpD patients are also likely to exhibit a similar brain biopterindeficit (or BH4 system deficit) that will result in the dysregulation ofneurotransmitters, such as dopamine, noradrenaline, serotonin and theglutamatergic system, and nitric oxide.

BpD patients treated with Li, have higher levels mRNA levels of GCH1,compared to patients not treated with Li. Also, BpD subjects treatedwith Li and who have the GCH1 “A” allele had lower GCH1 expression thanBpD subjects who were also treated with Li, but did not have the “A”allele. It is likely that Li upregulation of GCH1 is modulated by theGCH1 gene sequence, and Li treatment may be less effective, or havedifferent efficacy, in patients with the GCH1 “A” allele.

The present invention therefore involves the assay of GCH1 genotype,separately and/or in conjunction with assay of biopterin, BH4, otherpterins in plasma, other BH4 system measures, CSF, serum, urine, orother cells, tissues, organs, and fluid, and is useful in the screening,prediction, diagnosis and prognosis of psychiatric disorders, and fordefining treatments. In addition, treatments such as BH4, biopterin,other pterin species, Phe, lithium, or other treatments designed orknown or considered to increase biopterin or BH4 in persons with avariant GCH1 genotype (and thus to alleviate biopterin or BH4 systemdeficit, or prevent manifestation of a biopterin deficit in those atrisk), will be administered to provide a therapeutic or preventativeresponse or treatment in patients with the disorders or at-risk fordeveloping the disorders. The present invention will be useful fordetermination of risk, screening, early detection, diagnosis, andtreating psychiatric disorders including SZ, SaD, bipolar disorder(BpD), mood disorders and personality disorders, unipolar depressivedisorder, psychotic disorders, major depressive and other depressivedisorders, other affective disorders, attention deficit disorder,delusional disorder, anxiety disorders, obsessive compulsive disorder,paranoid schizotypal or schizoid personality.

Patients with Alzheimer's disease (AD) also have decreased biopterinand/or BH4 levels. The present invention may thus be useful fordetermination of risk, screening, early detection, diagnosis, andtreating AD and other neurological and neuropsychiatric andneurodegenerative disorders, including Pick's disease, Parkinson'sdisease, Huntington's disease, multiple sclerosis, Wilson's disease,Creutzfeldt-Jakob disease and other disorders of the central nervoussystem and the peripheral nervous system, and movement disordersincluding dyskinesias, dystonias and akathisias, and dementias, as wellas in intellectual or cognitive status impairment or decline. The samediagnostic and/or prognostic and/or treatment approaches would apply forthese disorders, as with psychiatric disorders such as those outlinedbelow for SZ and/or SaD and/or BpD.

The present invention also encompasses the screening, detection,prediction, diagnosis, early detection, prognosis, and/or treatment ofdisorders where a genetic or epigenetic (e.g., methylation and/oracetylation and/or ubiquitylation) difference affecting the GCH1 geneand/or its promoters, enhancers, suppressors and other regulatoryregions and/or RNA species (e.g., regulatory RNAs, such as miRNAs ornon-coding RNA), is present, such as a nucleotide variant, deletion,duplication, mutation, or change. The present invention further includesthe generation of genetic therapy treatments designed to provide GCH1DNA, RNA and/or GTPCH protein to subjects in need of treatment, and thatdo or do not carry deleterious variants. The present invention furtherincludes the generation of genetic therapy treatments designed toprovide BH4 system DNA, RNA, and/or protein(s) to subjects in need oftreatment. Subjects determined to be at-risk for development of apsychiatric, neuropsychiatric or neurological disorder, can includeinfants, children, adults, gametes, embryos, and/or fetuses, andassessment of risk status can be made from the individual, parental,and/or prenatal testing.

The present invention also encompasses kits for the screening,detection, prediction, diagnosis, early detection, prognosis, and/ortreatment of disorders where a genetic or epigenetic (e.g., methylationand/or acetylation and/or ubiquitylation) difference affecting the GCH1gene and/or its promoters, enhancers, suppressors and other regulatoryregions and/or RNA species (e.g., regulatory RNAs, such as miRNAs ornon-coding RNA), is present, such as a nucleotide variant, deletion,duplication, mutation, or change. As described herein, the presentinvention includes primers that may be used to identify the presence ofthe GCH1 alleles in humans.

Assessment of risk or screening is performed by genotyping subjects,along with, or without, a biopterin, BH4, BH4 system, other pterinsystem measurement (e.g., fasting plasma biopterin, GCH1 RNA, GTPCHprotein and/or neopterin measurement. Subjects with the rs10137071 GCH1“A/A” variant genotype alone or in conjunction with a “low” or “altered”BH4 system measure (e.g., biopterin), or the “A/G” or “A/A” genotype (orother DNA variants of the GCH1 gene) in conjunction with a measurementof “low” or “altered” biopterin (BH4, marker of low BH4, GCH1 RNA,and/or GTPCH protein), and/or other BH4 system measures, would beconsidered at an increased risk for developing a psychiatric disorder.

In addition, subjects with the variant genotype, low or alteredbiopterin, BH4, or BH4 system, altered GCH1 RNA, and/or GTPCH proteinlevels would be medicated with one or more of biopterin or BH4 or otherpterin species, or Phe or other treatment that increases BH4 (such aslithium) or inositol depletion, to alleviate the deficit, or treatmentto increase or supplement BH4 or biopterins, such as electrical brainstimulation or electroconvulsive therapy (ECT) or transcranial magneticstimulation (TMS).

Assessment of biopterin and/or BH4 and/or other pterin levels, alongwith GCH1 genotyping will also be used for screening or assessing thoseat-risk for developing a psychiatric, neuropsychiatric, or neurologicaldisorder, allowing for heightened monitoring, early detection, andearly, prodromal or prophylactic initiation of treatments. For thoseat-risk or with the disorders, treatment decisions can be made based ongenotyping and/or biopterin or BH4 level assay, and/or GCH1 RNA, GTPCHprotein level(s), and/or BH4 system measures.

The assay of GCH1 genotype, with or without biopterin or BH4 assay, canbe used to determine antipsychotic and/or mood stabilizer medication,and/or other treatment requirements. The efficacy of differentmedications and/or dosages will vary with genotype and biopterin and/orBH4 and/or GCH1 RNA and/or GTPCH protein level(s). For subjects with animpaired BH4 system (indicated by low biopterin, for example),treatments to increase BH4 can be used, such as BH4 supplementation,lithium treatment (patients treated with lithium have increasedbiopterin levels), phenylalanine treatment, or other treatments, such asECT, TMS, etc.

FIGS. 1A and B show the biosynthesis pathways of BH4 and its centralrole in the hydroxylation of the aromatic amino acids to the amineneurotransmitters, and its role as a cofactor in nitric oxide synthesis,and in the synthesis and release of neurotransmitters. In FIGS. 1A andB, GTPCH refers to GTP cyclohydrolase I, encoded by the GCH1 gene, PTPSrefers to 6-pyruvoyl-tetrahydropterin synthase, encoded by the PTS gene,SR refers to sepiapterin reductase, encoded by the SPR gene, DA refersto dopamine, NA refers to noradrenaline, 5-HT refers to5-Hydroxytryptamine, serotonin, NO refers to nitric oxide, and Glurefers to Glutamate.

As the amine neurotransmitter and glutamatergic systems and NO activityhave been implicated in the etiology of schizophrenia and affectivedisorders, a study of fasting plasma total biopterin (a measure of BH4)was performed. Study subjects included patients with BpD (n=27), SZ(n=154), SaD (n=59), and control subjects (n=37). For each patient, alifetime psychiatric diagnosis (using DSM-III-R criteria) was determinedbased on (a) clinical data collected from current and previousadmissions, and (b) diagnostic interviews. For control subjects, aninterview and internal scale were completed to determine the presence ofpersonal and/or family history for psychiatric, neurological, andmedical conditions. Demographics were collected for each of the subjectgroups, that included age, gender, ethnicity, years on neuroleptics(NL), and chlorpromazine equivalent dose (CPZE) (for the 21 days priorto blood draw), and the 4 groups (controls, BpD, SZ, SaD) were notsignificantly different for the variables of age, gender, ethnicitydistribution and body mass index. There was a significant difference inCPZ equivalence among patient groups, but no correlation between CPZequivalence (i.e., neuroleptic use) and biopterin levels was observed.

To test for differences in biopterin levels between the diagnosticgroups an initial GLM was used, with total biopterin as the primaryoutcome variable. This model, with the study group, the covariates ofgender, age, ethnicity, years of NL group, and CPZE group, 24 hourdietary Phe/protein ratio and plasma Phe, showed significant or nearsignificant main effects, or trends (on primary outcome variable offasting total biopterin), only for the variables of study group,ethnicity, and plasma Phe. A final model revealed only study group andplasma Phe as significant predictors of variation in total biopterinwith ethnicity approaching significance. Analysis of the main effectsrevealed that study group explained more than four times the amount ofvariation in log biopterin (Partial Eta Squared=0.112) than the varianceexplained by log Phe (Partial Eta squared=0.024). Pairwise comparisonsof log biopterin estimated marginal means (controlling for log Phe andethnicity) revealed significantly lower biopterin in SaD patients(p<0.001) and SZ patients (p<0.0001) than controls; specifically aplasma biopterin deficit of 34 percent in SZ patients and of 25 percentin SaD patients, when compared to the healthy control subjects, afterpartialling out the effects of potential confounds including gender,age, ethnicity, neuroleptic use history and dose of current use, 24-hourdietary phenylalanine/protein ratio (relevant to BH4 synthesis) andplasma phenylalanine (Phe) which stimulates BH4 synthesis. Of interest,BpD patients had significantly higher biopterin than SZ patients (meandifference±standard error=0.200±0.071, p<0.031), and were notsignificantly different from controls. SaD patients showed nosignificant difference from SZ or BpD patients.

A previous study of urine biopterin excretion showed no elevation in SZsubjects when compared to controls, suggesting that the plasma biopterindeficit results from a BH4 synthesis defect rather than increased urineexcretion.

In a further effort to explore the differences in plasma biopterindeficits between the psychiatric patient groups a second analysis wasperformed employing Lithium (Li) and mood stabilizer use as covariateson the model of plasma biopterin. In this new model, Li was found tohave a significant main effect on plasma biopterin. Post-hoc analysisadjusted for multiple testing showed that, in this model, all patientgroups had plasma biopterin levels significantly lower than controlsubjects. FIG. 2 illustrates the change in biopterin levels with Li as acovariate. This data suggests that Li treatment increases plasmabiopterin levels. The percent change in adjusted least squares means isshown for each patient group in FIG. 2 when lithium (yes/no) was addedto the final model (Li, Phe and ethnicity as covariates, on biopterin asthe outcome variable). Post-hoc Tukey test (adjusted) demonstrated thatSZ (p<0.0001), SaD (p<0.0001), and BpD(p=0.0179) differ from controls.No significant differences were found between patient groups. 89 percentof the BpD patients were treated with Li, approximately 50 percent ofthe SaD patients, and 2 percent of the SZ patients. The final modeldemonstrated a plasma biopterin deficit of 32 percent, 27 percent, and21.5 percent for SZ, SaD and BpD subjects respectively, when compared tocontrols.

DNA variants in a BH4 biosynthesis pathway gene thus appear to have acentral role in the etiology of SZ, via dysregulation of BH4 Synthesis,which would manifest as a biopterin deficit. The rate-limiting andinitial step in the de novo BH4 synthesis pathway is catalyzed by theenzyme GTP cyclohydrolase I (GTPCH (EC 3.5.4.16)) encoded by the GCH1gene, which maps to chromosome 14q22. The present invention thusconsidered the genotype data for a biallelic nucleotide variant (−959ntG to A: rs10137071, NCBI dbSNP database) in the GCH1 gene promotersequence, previously described as having association with bipolardisorder. The −959nt G/A variant lies in the 5′ upstream promoter ofGCH1, and has a reported heterozygosity of 0.46 (23). Testing forassociation of this common variant with SZ and SaD in a mixed US sampleof 174 subjects (86 SZ subjects, 42 SaD subjects and 46 controlsubjects) and testing for an association of GCH1 with the biopterindeficit in subject groups demonstrate that a GCH1 gene variant increasesrisk of major psychiatric disorders, and is associated with lowbiopterin levels in psychiatric patients. The results support a geneticbasis for BH4 deficiencies in psychiatric disorders.

For each patient subject, a lifetime psychiatric diagnosis (usingDSM-III-R criteria) was determined based on (a) clinical data collectedfrom current and previous admissions, and (b) diagnostic interviewsconducted by the research team. For control subjects, a questionnairewas completed to determine the presence of personal and/or familyhistory for psychiatric illness. Control subjects had no history ofpsychiatric disorders, although 1 control subject had previouslyexperienced mild, age-related depression, but had not sought treatment.After complete description of the study to the subjects, writteninformed consent was obtained.

Genomic DNA was extracted from whole blood using standard methods(Gentra Systems, Inc). Primers were utilized to amplify a 351 bp genomicregion spanning the G/A variant in a 35 cycle PCR reaction. Therestriction enzyme BsrD1 recognizes and cleaves the A allele resultingin the formation of a 191/160 bp doublet. Digested products werevisualized following electrophoreses. Subjects homozygous for the Gallele were genotyped by the presence of a single 351 bp band,heterozygous subjects have two visible bands, the 351 bp G allele andthe 191/160 bp A allele, while A/A subjects have only the 191/160 bpdoublet.

Sample preparation and assay for plasma total biopterin has previouslybeen reported and measurement of plasma Phe (assayed due to its knownrole in the regulation of BH4 synthesis) has also been reported. Testsfor Hardy Weinberg equilibrium, ethnic distribution of study groups, andinitial model of association between GCH1 genotypes (A/A, G/A, G/G) anddiagnostic group (SZ, SaD, C) were performed using exact tests. Under arecessive model (testing the difference between the minor allele (A)homozygotes versus G/A heterozygotes and major allele (G) homozygotes),logistic regression was employed to test for association withpsychiatric group status. Wald chi-squares and odds ratios (OR) withWald 95% Confidence Intervals (Wald CI) were reported with and withoutethnicity as a covariate.

Functional analyses of the GCH1 genotype using logistic regression andgeneral linear models were conducted on a subset of subjects with plasmabiopterin data available. For logistic models, dependent variables werepsychiatric status and independent variables were GCH1 genotype,biopterin level and the interaction of GCH1 genotype with biopterinlevel. Ethnicity was used as a covariate in the initial model.

A general linear model was used to test the effect of GCH1 genotype,psychiatric disorder status and their interaction on biopterin level. Asthe plasma concentration of Phe in fasting subjects has a significanteffect on plasma total biopterin levels, and Phe was employed as acovariate to measure the effect of GCH1 genotype on biopterin level.Post-hoc significance testing was reported using a Tukey-Krameradjustment for multiple comparisons in all logistic and linear models.Statistical analyses were conducted using SAS 9.1.2 (FREQ, LOGISTIC andGLM Procedures, SAS Institute Inc, 2004) and exact tests for HardyWeinberg equilibrium were calculated using R with the genetics library(R: A language and environment for statistical computing. R Foundationfor Statistical Computing, Vienna, Austria, 2006, version 1.2.0, 2005).

Exact tests for Hardy-Weinberg equilibrium demonstrated equilibrium forthe patient (n=128, p=0.1534), and control groups (n=46, p=0.1299).Ethnic distributions of SZ, SaD, and control samples were notsignificantly different (Fisher exact test, n=174, p=0.4234). Overallfrequencies: Patient group-42.2% Caucasian, 39.8% African-American,18.0% Hispanic; Control group-56.5% Caucasian, 30.4% African-American,13.1% Hispanic. Subject group demographics are presented in Table 1below. Exact counts (and %) of GCH1 genotypes are shown for each of thestudy and ethnic groups. The A/A genotype was significantly associatedwith both SZ and SaD. Significance was observed in the African-Americangroup and a trend toward significance in the Hispanic group. Afteradjusting for ethnicity, the associations detected in the recessivemodel remained significant in all groups tested.

TABLE 1 Diagnostic and Ethnic Group Comparison of GCH1 Genotype. WaldChi-Square (p=) GCH1 Genotype Recessive Model Subjects (n = 174) A/A G/AG/G Model (adjusted) SZ (n = 86) 30 (34.9) 34 (39.5) 22 (25.6) 0.00240.0057 Controls (n = 46) 4 (8.7) 27 (58.7) 15 (32.6) SaD (n = 42) 12(29) 21 (50) 9 (21) 0.0216 0.0288 Controls (n = 46) 4 (8.7) 27 (56.7) 15(32.6) Patients (n = 128) 42 (32.8) 55 (43) 31 (24.2) 0.0033 0.0066Controls (n = 46) 4 (8.7) 27 (58.7) 15 (32.6) African-American (n = 65)Patients (n = 51) 25 (49) 21 (41.2) 5 (9.8) 0.0312 — Controls (n = 14) 2(14.3) 12 (85.7) 0 (0) Caucasian (n = 80) Patients (n = 54) 7 (13) 28(51.8) 19 (35.2) n/s — Controls (n = 26) 2 (7.7) 12 (46.15) 12 (46.15)Hispanic (n = 29)* Patients (n = 23) 10 (43.5) 6 (26.1) 7 (30.4) 0.0676— Controls (n = 6) 0 (0) 3 (50) 3 (50) *In the Hispanic group (n = 29),a valid maximum likelihood estimate could not be calculated and a FisherExact Test was performed. n/s, not significant

In an initial analysis, an association between GCH1 genotype (A/A, A/Gand G/G) and SZ (Fisher exact test, p=0.0027), and SaD (Fisher exacttest, p=0.05) was revealed. When patient groups were combined, asignificant association remained (n=174, Fisher exact test, p=0.0038).Most notably, under a recessive model the inventors found a highlysignificant association between the A/A genotype and SZ (Waldchi-square=9.2, df=1, p=0.0024) and significant association with SaD(Wald chi-square=5.3, df=1, p=0.0216). When the patients were combined(patients n=128 and controls n=46), a highly significant associationremained (Wald chi-square=8.6, df=1, p=0.0033). The odds ratios (OR) ofhaving a SZ or SaD diagnosis amongst the homozygous A/A population were5.6, and 4.2 respectively. Combining the patient groups yielded an OR of5.1.

Although subject ethnicities were not different between patient andcontrol groups, assuming a degree of stratification in the mixed USpopulation of the study, the inventors added ethnicity as a covariate inthe model. The resulting OR's adjusted for ethnicity were 5.0 (p=0.0057,95% Wald CI: 1.601-15.886), for SZ and 4.1 for SaD (p=0.0288, 95% WaldCI: 1.157-14.389), 4.7 combined patient group (p=0.0066, 95% Wald CI:1.54-14.48). Thus, the A/A genotype confers a highly significantincreased risk of having a psychiatric disorder.

The association of the A/A genotype with psychiatric disorders differedamong ethnic groups within the study sample (see Table 1). In patientand control subject groups, the highest prevalence of the A/A genotypewas found in the African-American subjects (patients 49.0%, controls14.3%). Further association testing performed within the specific ethnicgroups illustrated a significant association of the A/A genotype withdiagnostic group in the African-American subset of patients, which was atrend in the Hispanic subjects. The A/A genotype was 69% more frequentin patients than controls in the Caucasian group.

The biological relevance of the GCH1 genotype was initially investigatedthrough analysis of plasma biopterin and genotype interaction. Theplasma biopterin level is significantly lower in SZ when compared tocontrol subjects. There is seen in FIG. 3, an individual subject'sranked plasma biopterin levels and GCH1 genotype within each diagnosticgroup displayed by Study Group, Plasma Biopterin level and GCH1Genotype. Subjects are ranked by biopterin level (increasing left-right)within their study group, illustrating both the decreased biopterinlevels in the SZ (n=52) and SaD (n=32) subject groups compared to thecontrol group (C, n=32), and the relative preponderance of A/A genotypeamong patients with the lowest biopterin levels. Plasma biopterin levelsof the SZ and SaD subjects were not significantly different from eachother, however both were significantly different from control subjects(p<0.01 for both SZ and SaD groups). BpD patients also had significantlylower biopterin than controls when biopterin levels were adjusted forthe effects of Lithium treatment.

Table 2 below shows descriptive statistics for biopterin level (andstandard deviations (SD)) for each subject group, separated by GCH1genotype. Biopterin values and GCH1 Genotypes for subject groups(Patient and Control). The mean biopterin levels (nM) and standarddeviation (SD) within each subject group is shown for each GCH1genotype. Final biopterin values were adjusted by fasting Phemeasurements.

TABLE 2 Mean Biopterin Adjusted Mean Subjects n (nM) SD Biopterin (nM)Patients 84 AA 29 10.7 3.9 10.7 AG 35 10.8 3.4 10.8 GG 20 13.6 5.5 13.9A/A A/G 64 10.7 3.6 10.8 G 20 13.6 5.5 13.9 Controls 32 AA 3 17.5 6.517.2 AG 22 15.1 3.7 15.0 GG 7 15.1 5.2 14.7 AA/AG 25 15.4 4.0 15.2 G 715.1 5.2 14.7

The plasma biopterin values and GCH1 Genotypes for the subject groupdata is plotted in the FIG. 4 for patients (Panel A) and controls (PanelB). Plasma biopterin values (nM) (unadjusted for fasting phenylalaninelevels) were plotted for patient subject genotype (Panel A), A/A (n=29),G/A (n=35) and G/G (n=20) and for control subject genotypes (Panel B),A/A (n=3), G/A (n=22) and G/G (n=7). No significant difference inbiopterin level was observed between A/A or G/A genotypes in the patientgroup, however both the A/A and G/A patient groups were significantlydifferent from the G/G patients (Tukey adjusted p-values for multiplecomparisons: p=0.0165 and p=0.0168, respectively). Patient subject Aallele carriers were combined for further analysis (patient A allelecarriers-blue box-plots). No significant difference in biopterin levelwas observed among the control genotypes (control A allele carriers—pinkbox-plots). Panel C shows patient A allele carriers (n=64) havedecreased biopterin levels compared to homozygous G/G patients (n=20),and to all control subject groups (lines indicate Tukey adjustedp-values for multiple comparisons: ** p<0.05, *** p<0.0001). Box plotsdisplay the median (horizontal line in box), first (Q1) and third (Q3)quartiles (ends of the boxes). Bars outside the boxes represent theextreme values within 1.5 times the inter-quartile range (IQR) from theupper or lower quartile. Points at a greater distance from the medianthan 1.5 times the IQR are plotted individually as small circles.

A logistic regression analysis was performed using all subjects withboth plasma biopterin level and GCH1 genotype (patient group n=84;control group n=32) and the interaction between plasma biopterin andGCH1 genotype was tested for the outcome variable “study group,”controlling for ethnicity. Although the interaction between A/A genotypeand biopterin on study group (Patient, Control) was not significant, andpost-hoc comparisons did not show a significant difference betweenbiopterin levels of patient subjects with the A/A and G/A genotypes, themean biopterin levels of both A/A and G/A patient groups weresignificantly different to mean biopterin for G/G patients (A/Ap=0.0165, A/G p=0.0168) and so the A/A and G/A patient groups werecombined to form an A allele carrier group.

The interaction of GCH1 A allele and low biopterin level, was found tobe a significant predictor of diagnostic group (patient or control)(Wald chi-square=4.8, df=1, p=0.0286). Ethnicity was not a significantpredictor of study group (Wald chi-square=0.1, df=1, p=0.8962) and wasthus removed from the final model (adjusted p-value of final modelinteraction, p=0.0276, model concordance at 79.8%). Table 3 below showsparameters from the logistic models. Logistic regression was employed totest the interaction between plasma biopterin level and GCH1 genotype,as a significant predictor of study group (patient or control, n=116),using ethnicity as a covariate in the initial model.

TABLE 3 GCH1 A allele Status and Biopterin Level as a Predictor of StudyGroup. Wald Standard Chi- Pr > Parameter DF Estimate Error Square ChiSqInitial Model ^(a) Intercept 1 3.2294 0.8042 16.1268 <.0001 GCH1 Aallele 1 1.4525 0.8122 3.1982 0.0737 Biopterin (n/M) 1 −0.1693 0.053510.0163 0.0016 Biopterin * GCH1 A 1 −0.1179 0.0537 4.8213 0.0281 alleleCaucasian vs African 1 −0.0178 0.3400 0.0028 0.9582 American Hispanic vsAfrican 1 0.1531 0.4509 0.1153 0.7342 American Final Model ^(b)Intercept 1 3.2055 0.7970 16.1774 <.0001 GCH1 A allele 1 1.4206 0.79703.1772 0.0747 Biopterin (n/M) 1 −0.1693 0.0536 9.9888 0.0016 Biopterin *GCH1 A 1 −0.1180 0.0536 4.8531 0.0276 allele ^(a) Overall model:Likelihood Ratio chi-square = 22.9, df = 5, p = 0.0004, 80.1%Concordance rate ^(b) Overall model: Likelihood Ratio chi-square = 22.7,df = 3, p < .0001, 79.8% Concordance rate

Further supporting this detected interaction, tests showed that GCH1allele status is itself a significant predictor of biopterin level. Asfasting phenylalanine (Phe) levels are known to be predictors of plasmabiopterin levels, adjustments were made for Phe. While the GCH1 alleledid not have a significant main effect on plasma biopterin, levelswithin the patient group were significantly lower in patients with the Aallele compared to those without (Tukey adjusted p-values for multiplecomparisons, p=0.0208). In control subjects no significant difference inbiopterin levels for GCH1 allele status was detected (Tukey adjustedp=0.9924) (see FIG. 3, which shows for unadjusted biopterin levels andsignificance testing between subject groups).

The sequence of the GCH1 promoter in which the GCH1 variant of thepresent invention lies is seen in FIG. 5. Also seen in FIG. 5 are thesequences for the forward and reverse primers that may be used toidentify the GCH1 alleles of the present invention. The nucleotidesequence of the 351 bp DNA product that is amplified using the primerpair (green) is shown. The published SNP rs10137071 (the SNP rs10137071is published as a C/T variant as the GCH1 gene is transcribed in thereverse DNA strand), also referred to as the −959nt G/A variant, isunderlined in the sequence (red) and the two possible alleles of thepresent invention are shown. Human subjects can be G/G, A/G or A/A atthe relevant nucleotide position.

GCH1 mRNA transcripts are lower in peripheral tissues from A allelepatient carriers. Whole blood leukocytes collected from subjects with SZ(both medicated and non-medicated SZ subjects), and BpD, and alsoethnicity and gender matched control subjects, and global leukocyte geneexpression were measured using Affymetrix microarrays. For each subjectrecruited, a 15 ml blood sample was collected. Immediately after bloodcollection, leukocytes were isolated by lysis of red cells,centrifugation and washing (Qiagen). Purified leukocytes were stored at−70° C. prior to RNA extraction (and are stable for periods of >1 year).Total RNA was extracted using RNeasy columns (Qiagen), and quantified byUV spectrometry using RNA standards for normalization or using anAgilent Bioanalyzer. Subjects were also genotyped for the GCH1 promotervariant, as described herein.

Only RNA samples with good quality ribosomal RNA, and satisfactory O.D.260/280 ratios were processed to completion. 8 μg of total RNA wasemployed as a cDNA synthesis template, using an oligo-dT primer andReverse Transcriptase (RT) enzyme, according to standard Affymetrixprotocols. Purified cDNA, was then used as a template to generate biotinlabeled cRNA (Enzo). cRNA samples were quantified and stored at −70° C.prior to fragmentation. 20 ng of each fragmented cRNA product washybridized to an Affymetrix TEST3 array to check sample quality and thenhybridized to an HU133 plus 2.0 array (containing over 40,000transcripts).

Initial comparison of GCH1 leukocyte mRNA levels between SZ, BpD, andcontrol subjects showed that GCH1 was downregulated in patient groups,which is consistent to that found in the CNS. SZ patients with the Aallele has significantly lower plasma biopterin levels when compared tocontrols, while G/G SZ patients had similar levels to controls (andthere were no differences between any of the control subject genotypes).As a result, differences in GCH1 transcript levels in A allele patientswere tested as compared to G/G patients. Referring to FIG. 6,significantly lower leukocyte GCH1 expression was found in the SZ Aallele group (n=12) when compared to SZ G/G subjects (n=7), which isconsistent with plasma biopterin data obtained from this subject group.Data in FIG. 6 are natural log (1n) normalized via RMA, where the firstpanel illustrates GCH1 expression is significantly lower in peripheralleukocytes from A allele SZ patients compared to SZ G/G patients(p=0.024). The second panel of FIG. 6 illustrates that GCH1 expressionis lower in A allele BpDpatients compared to G/G BpD patients (p=0.2).The box plots display the mean (+), median (horizontal line in box),first (Q1) and third (Q3) quartiles (ends of the boxes). Analysis of 17BpD subjects also demonstrated lower levels of GCH1 in A allele carriers(n=9), when compared to G/G BpD patients (n=8), although this result wasnot significant.

Ten of the BpD subjects employed for this analysis had been treated withlithium, seven had not (16 of the 17 BpD subjects were also receivingneuroleptics). Subjects were therefore separated for both genotype andLi treatment groups, with the means calculated and plotted in FIG. 7,which illustrates mean log expression for BpD subjects without Litreatment (solid line) with the AA (n=2), A/G (n=4) and G/G (n=4)genotypes. Log mean expression for BpD subjects with Li treatment(dashed line) with the A/A (n=0), A/G (n=3) and G/G (n=4) genotypes.Although the results were not significant (likely due to small number ineach group), the data was striking and consistent with all previousresults, i.e., levels of GCH1 in the BpD Li untreated group (lowest inthe A/A subjects and highest in the G/G BpD subjects) had a similarexpression profile to the plasma biopterin levels in SZ subjects (seeFIG. 4). In addition, subjects treated with Li had higher expressionlevels than subjects without Li treatment. Plasma biopterin levels showthat Li-treatment likely increases plasma biopterin levels, which islikely accomplished via increased GCH1 expression.

The data thus suggests that subjects with the GCH1 BpD-associatedgenotype will have downregulated GCH1 mRNA (in both the periphery andCNS) that alters BH4 biosynthesis. The resultant BH4 deficit will thuscontribute to BpD susceptibility, likely via altered CNSneurotransmission. The data also suggests that for Li treated patients,the biopterin deficit can be alleviated via increased GCH1 expression,which is supported by a similar GCH1 mRNA rise observed in the rat brainfollowing Li-induced inositol depletion (via Li administration). Li hasa major effect on plasma biopterin levels, thus suggesting that theincreased biopterin levels in BpD subjects is due, in part, to Liupregulating GCH1 transcription and subsequently BH4 biosynthesis. Litreatment may thus be less effective in subjects with the BpD associatedallele, and the data supports targeting of treatment for BpD based onGCH1 genotype.

With respect to the prediction of risk and/or screening, the presentinvention provides for the assay of GCH1 genotype for the presence of aGCH1 variant as a method for prediction of risk for SZ or SaD or apsychiatric disorder, neuropsychiatric disorder, or neurologicaldisorder. The present invention also provides for the assay of GCH1genotype for the presence of a GCH1 variant, in conjunction with theassay of BH4 system measures (e.g., plasma biopterin assay), as a methodfor prediction of risk and/or screening for SZ, SaD, or BpD, or otherpsychiatric disorders, neuropsychiatric disorders, or neurologicaldisorders.

With respect to diagnosis, the present invention provide for the assayof GCH1 genotype for the presence of a GCH1 variant, in conjunction withthe assay of BH4 system measures (e.g., plasma biopterin assay), as amethod for diagnosis of SZ or SaD, or BpD or a psychiatric disorder,neuropsychiatric disorder, or neurological disorder. The presentinvention also provides for the assay of GCH1 genotype for the presenceof a GCH1 variant, as a method for diagnosis of SZ or SaD, or otherpsychiatric disorders, neuropsychiatric disorders, or neurologicaldisorders.

With respect to diagnosis incorporating symptoms, the present inventionprovides for the assay of GCH1 genotype for the presence of a GCH1variant, in conjunction with assessment of specific symptoms andsymptoms types, and in conjunction with the assay of BH4 system measures(e.g., plasma biopterin assay), as a method for diagnosis of SZ or SaD,or BpD or a psychiatric disorder or neuropsychiatric disorder, and/orthe analysis of symptoms for those disorders. The present invention alsoprovides for the assay of GCH1 genotype for the presence of a GCH1variant, in conjunction with assessment of specific symptoms andsymptoms types, as a method for diagnosis of SZ or SaD, or otherpsychiatric disorders, neuropsychiatric disorders, or neurologicaldisorders.

With respect to prediction of prognosis, the present invention providesfor the assay of GCH1 genotype for the presence of a GCH1 variant, inconjunction with the assay of BH4 system measures (e.g., plasmabiopterin assay), as a method for prediction of prognosis of SZ or SaD,or BpD or a psychiatric disorder or neuropsychiatric disorder. Thepresent invention also provides for the assay of GCH1 genotype for thepresence of a GCH1 variant as a method for prediction of prognosis of SZor SaD, or other psychiatric disorders, neuropsychiatric disorders, orneurological disorders.

With respect to the assessment of patient treatment benefit, the presentinvention provides for the assay of GCH1 genotype for the presence of aGCH1 variant, in conjunction with the assay of BH4 system measures(e.g., plasma biopterin assay), as a method for defining those patientswith a psychiatric disorder, neuropsychiatric disorder, or neurologicaldisorder (e.g., SZ, SaD, BpD) who would benefit from treatment designedto normalize their BH4 system levels, or prevent a potential biopterinor BH4 system deficit in those at-risk. The present invention alsoprovides for the assay of GCH1 genotype for the presence of a GCH1variant, as a method for defining those patients with a psychiatricdisorder, neuropsychiatric disorder, or neurological disorder (e.g., SZ,SaD, BpD) who would benefit from treatment designed to normalize theirBH4 system levels and/or improve their symptoms.

With respect to defining at-risk subjects, the present inventionprovides for the assay of GCH1 genotype for the presence of a GCH1variant as a method for defining those at risk for SZ or SaD or otherpsychiatric, neuropsychiatric, or neurological disorder. The presentinvention also provides for the assay of GCH1 genotype for the presenceof a GCH1 variant, in conjunction with the assay of BH4 system measures(e.g., plasma biopterin assay), as a method for defining those at riskfor SZ, SaD, or BpD, or other psychiatric disorders, neuropsychiatricdisorders, or neurological disorders.

With respect to defining at-risk subjects who would benefit fromprophylactic treatment, the present invention provides for the assay ofGCH1 genotype for the presence of a GCH1 variant, in conjunction withthe assay of BH4 system measures, as a method for defining those at riskfor development of SZ or SaD or BpD or a psychiatric disorder,neuropsychiatric disorder, or neurological disorder who would benefitfrom treatment designed to normalize their BH4 system levels and/orimprove their symptoms. The present invention also provides for theassay of GCH1 genotype for the presence of a GCH1 variant as a methodfor defining those at risk for development of a psychiatric,neuropsychiatric or neurological disorders, including SZ or SaD, andthose who would benefit from useful treatments designed to normalize orimprove their BH4 system levels, improve their symptoms, and/or benefitfrom Li, neuroleptic(s), and/or other antipsychotic or mood stabilizers,and/or treatments (e.g., administration of electrical stimulation and/orelectroconvulsive therapy (ECT), transcranial magnetic stimulation(TMS), electrical brain stimulation, deep brain stimulation, and/orinositol depletion treatments).

With respect to the treatment and prevention of disorders, the presentinvention provides for the treatment of subjects with, or who are atrisk of developing, SZ or SaD and/or a psychiatric disorder,neuropsychiatric or neurological disorder, who either carry a GCH1variant and/or a BH4 system gene variant and/or a variant in anothergene that can lead to BH4 system deficit or alteration, or who have (orare at risk for developing) a fasting or non-fasting biopterin level(e.g. plasma biopterins) and/or BH4 and/or pterin levels, and/or GCH1RNA and/or GTPCH protein that lie in the range defined as “low” or“altered” or is different from the mean (or median, mode, normal range,expected value, etc.) compared to controls, by treatment with usefultreatments. Treatments will be used as a way to boost and/or alter BH4levels, biopterin levels, and/or affected BH4 system component levels inthose in need of BH4 system supplementation and/or normalization, withand/or at-risk of developing a disorder. Such treatment(s) can be usedeither alone or in conjunction with other “useful treatments” whichinclude, but are not limited to: treatment with pterin(s) and/or othermolecules, including BH4 and/or biopterin and/or dihydrobiopterin and/orsepiapterin and/or sapropterin dihydrochloride. Other medications thatmay improve BH4 system status or that can be used in conjunction withsuch treatments may include neuroleptic or other psychotropicmedications (e.g., Phenothiazines, Chlorpromazine, Fluphenazine,Perphenazine, Prochlorperazine, Thioridazine, Trifluoperazine,Butyrophenones, Haloperidol, Droperidol, Pimozide, Clozapine,Olanzapine, Risperidone, Quetiapine, Ziprasidone, Aripiprazole,Bifeprunox; norclozapine (ACP-104), Symbyax, Tetrabenazine, andlithium), ECT, TMS, and other like medications. Such treatment(s) may beprovided to those with SZ, SaD, BpD, or BH4-responsive PKU,hyperphenylalanenemia (Phe and/or aspartame and/or Phe-containingpolypeptides will not be used for hyperphenylalaninemia), and/ordystonias. Such treatments will also be used for other psychiatricand/or neuropsychiatric disorders and/or neurological disorders.

With respect to the treatment of patients with useful treatmentsdesigned or used to normalize BH4 and/or BH4 system levels, the presentinvention also provides for the treatment of SZ, SaD, or BpD who carry aGCH1 variant (or a BH4 system gene variant), in conjunction with afasting or non-fasting biopterins (e.g., plasma biopterins), BH4, BH4system measures, pterin levels, GCH1 RNA and/or GTPCH protein, that liein the range defined as “low” or “altered” compared to controls, withtherapeutic amounts of useful treatments. The present invention alsoprovides for the treatment of SZ, SaD, or BpD (or other psychiatricdisorders, neuropsychiatric disorders, or neurological disorders in needof treatment), in subjects who carry a GCH1 gene (or a BH4 system gene)variant, by treatment with therapeutic amounts of useful treatments. Itshould also be recognized by those of skill in the art that the presentinvention may be used to determine the presence of a GCH1 G/G genotype,and any BH4 system surplus, such as high BH4 and/or bopterin, that mayfactor into diseases symptoms, disease risk, and/or the applicabletreatment response.

With respect to the treatment of at-risk subjects with useful treatmentsdesigned or used to normalize BH4, the present invention provides forthe treatment of those at risk of developing SZ, SaD, or BpD, or otherpsychiatric disorders or neuropsychiatric disorders or neurologicaldisorders (or other disorders in need of treatment) who carry a GCH1variant (or a BH4 system gene variant) in conjunction a “low” or“altered” BH4 system and/or with a fasting or non-fasting biopterinlevels (e.g., plasma biopterins), BH4 levels, and/or pterin levels, aswell as GCH1 RNA and/or GTPCH proteins that lie in the range defined as“low” or “altered” or is different from the mean (or median, mode,normal range, expected value(s) etc.) compared to controls and/or otherpatient groups, by treatment with useful treatments. The presentinvention also provides for the treatment of those at risk of developingSZ or SaD or BpD (or other disorders in need of treatment) who carry aGCH1 variant (or a BH4 system gene variant), by treatment with usefultreatments. Subjects determined to be at-risk for development of apsychiatric, neuropsychiatric or neurological disorders can includegametes, embryos, and/or fetuses, and assessment of risk status can bemade from parental and/or prenatal testing.

With respect to prevention and/or treatment using gene or proteintherapies, the present invention provides for the treatment of thosewith, or at risk of developing, SZ or SaD or a psychiatric,neuropsychiatric, or neurological disorder (or other disorders in needof treatment) who carry a GCH1 variant (or a BH4 system gene variant),either alone or in conjunction with a fasting or non-fasting biopterins(e.g. plasma biopterins) and/or BH4 and/or pterin levels, and/or GCH1RNA and/or GTPCH protein that lie in the range defined as “low” or“altered” or different from the mean (or median, mode, normal range,expected value, etc.) compared to controls, by treatment withtherapeutic DNA and/or RNA molecules to cells, tissues, or organs, orlocally or systemically (including injection, intravenously, orally,suppository, intranasally, intracerebrally, intrathecal, parenterally,or by infusion), or by administration to isolated cells, tissues,organs, fluids, etc. Therapeutic DNA and/or RNA molecules include, butare not limited to, sequences related to those of GCH1 (such as forexample sequence containing the −959nt G to A: rs10137071, “G” allele),other BH4 system genes, and/or related genes, and can be introduced byusing vectors, viral particles, capsules, liposomes, and othercontainers or delivery methods, or by binding to other molecules orparticles, surfaces, or materials. Therapeutic DNA and/or RNA moleculescan also be introduced to sperm, eggs, embryos, fetuses, stem cells, orother cells. Additionally, in conjunction with these procedures, one ormore other useful treatments may also be used.

With respect to imaging correlates in the screening, detection,diagnosis, prognosis, definition of at-risk, and assessment of treatmentresponse, the present invention provides for the assay of GCH1 genotype(or assay of a BH4 system gene variant), altered biopterin level, and/orBH4 system measures (e.g., biopterin), for those with or at risk ofdeveloping a psychiatric, neuropsychiatric, or neurological disorder orother disorder, and the determination use of MRI, MRS, fMRI, PET, SPECT,CT, or other imaging technologies and methods to assess CNS, peripheral,structural, biochemical activity, physiological, and/or otherdifferences or changes in subjects.

With respect to gene or protein expression in the screening, detection,diagnosis, prognosis, definition of at-risk, and assessment of treatmentresponse, the present invention provides for the assay of GCH1 genotype(or of a detected BH4 system gene variant) and/or assay of the BH4system or BH4 system measures, for those with or at risk of developing apsychiatric, neuropsychiatric, or neurological disorder, and the use ofany RNA (including miRNA) and/or protein and/or peptide expressionmeasurement, such as microarray analysis, mass spectrometry analysis,protein microarray analysis or electrophoresis analysis, and can be usedfor disease and/or symptom detection, diagnosis, prognosis, definitionof at-risk, determination of treatment, assessment of treatmentresponse. Expression analysis in any tissue, and/or organs and/or cells,from a subject or animal model could be employed. Expressionmeasurements, profiling, and detection methods are included to assessCNS, peripheral, structural, biochemical activity, physiological, and/orother differences or changes in subjects.

With respect to the detection, diagnosis and medication of subjectshaving BH4 system gene mutations and variants and alterations, thepresent invention provides for the screening, determination of at-risk,detection, diagnosis, prognosis, determination of treatment andtreatment of disorders, including whether there is a genetic orepigenetic (e.g., methylation, acetylation, and/or ubiquitylation)difference affecting the gene(s), as well as their promoters, enhancers,suppressors and other regulatory regions, regulatory RNAs (such asmiRNAs), such as whether there is a nucleotide variant (e.g., a G/Anucleotide substitution or a nucleotide deletion), or copy numberpolymorphism and/or duplication, deletion or other mutation or change,whether newly detected or previously known, such as for GCH1, NURR1, GSK(including GSK 3 beta and alpha), IMPase, AKT1, AKT/PKB, AKT2, AKT3 orIPP mutations and variants, and for other BH4 system genes.

With respect to the generation of, and/or breeding with, and/or use of aGCH1 transgenic animal model and/or animal model with a targeteddisruption of the GCH1 gene, the present invention provides for researchinto the etiology and pathogenesis of, as well as the development ofmediations and treatments, for neuropsychiatric, and neurologicaldisorders, including SZ, SaD and BpD.

What is claimed is:
 1. A method of treating a human subject for neuropsychiatric disorder, comprising the steps of: determining a GTP cyclohydrolase I (GCH1) −959 nt G/A genotype of the subject; determining the levels of at least one of total biopterins, BH4, and dihydrobiopterin of the subject; and treating the human subject for the neuropsychiatric disorder based on the genotype or biopterin level by administering a therapeutic amount of a BH4 supplement if the levels of at least one of total biopterins, BH4, and dihydrobiopterin of the human subject are below a predetermined threshold and if the human subject has an A allele of the GCH1 −959nt G/A genotype.
 2. The method of claim 1, wherein the neuropsychiatric disorder is cognitive impairment.
 3. The method of claim 2, wherein the BH4 supplement is sapropterin dihydrochloride.
 4. A method of treating a human subject for a neuropsychiatric disorder comprising the step of administering a therapeutic amount of a BH4 supplement.
 5. The method of claim 4, wherein the neuropsychiatric disorder is cognitive impairment.
 6. The method of claim 5, wherein the BH4 supplement is sapropterin dihydrochloride. 